Antithrombin III or heparin cofactor is the major plasma inhibitor of the blood coagulation proteases. Heparin greatly enhances the rate of protase inactivation without altering the stoichiometry of the reaction. This system has been most extensively studied with the protase thrombin. The increase in rate of thrombin inactivation appears to result from the formation of a noncovalent thrombin-antithrombin-heparin ternary complex. Consistent with this model, we have shown that specific chemical modification of essential lysine or typtophan residues in antithrombin III blocks heparin binding and the heparin accelerated inactivation of thrombin, without altering the intrinsic rate of thrombin inhibition. Peptide mapping and amino acid sequence analysis will be used to identify these essential residues in the primary structure of antithrombin. To further define the structural requirements for heparin interaction with antithrombin we propose to use limited proteolysis of antithrombin to generate functional heparin binding fragments. These fragments will be detected using heparin derivatives containing fluorescent or chromophoric labels. Antibodies directed against the antithrombin heparin binding domain will be produced and their effects on antithrombins functional properties will be evaluated to determine if they block heparin binding and heparin cofactor activity. Site specific chemical modification of tryptophans will be used to assess the contribution of individual residues to heparin binding and to reaction with thrombin and with Factor Xa. Additionally, we intend to use hydrodynamic methods to detect and quantitate formation of specific complexes of heparin with antithrombin, thrombin, and Factor Xa. These studies should allow direct demonstration of the putative noncovalent protease-antithrombin-heparin tenary complex.